Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules

Background

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson’s disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown.

Results

By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca²⁺ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains.

Conclusions

Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-729) contains supplementary material, which is available to authorized users.     

In vitro assessment of some sperm function following exposure to levonorgestrel in human fallopian tubes

Background  

The mechanism of action of levonorgestrel (LNG) as emergency contraception (EC) remains a subject of debate and its effect on sperm function has been only partially explained. The aim of this study was to assess whether LNG at a similar dose to those found in serum following oral intake for EC could affect spermatozoa when exposed to human fallopian tubes in vitro.     

Structural investigations on novel porin, OmpAb from Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen and known to cause nosocomial infections especially in ICUs of hospitals. We have previously reported that the novel outer membrane protein, OmpAb from Acinetobacter baumannii is a transmembrane porin and plays an important role in transport of small molecules, like antibiotics across the membrane. In the present study we report the N-terminal sequence, Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis of OmpAb and structural investigations using UV-Vis absorption, circular dichroism (CD), and fluorescence on OmpAb. SDS-PAGE results suggest that OmpAb is actually a "heat modifiable monomer" and is of 37 kDa at room temperature. Secondary structure of OmpAb is being done for the first time that showed predominantly beta-sheet structure (68%), a feature characteristic of porins. Using N-Bromosuccinimide (NBS) as oxidizing agent, the total number of tryptophans in OmpAb is estimated to be four. The present results indicate that out of the four, two tryptophans seem to be located in the integral part of the membrane, perhaps periplasmic/membrane-bound while the other two tryptophans are exposed to the solvent. We followed the fluorescence emission using conventional 280 nm and selective 305 nm excitation (established by us earlier) to explore the environment of four tryptophans in OmpAb. Emission results using selective excitation of 305 nm revealed local conformational changes of those "tryptophans which are on the surface". On urea denaturation and pH dependent denaturation there is a loss of beta-sheet structure by more than 70%, this is concomitant with the increase in fluorescence intensity and red shift in lambda(max, em). As reflected by CD spectral data, we also found that OmpAb is fairly stable like other porins up to 70 degrees C. As there are no reports on the structural aspects of any outer membrane proteins of Acinetobacter baumannii, results presented here on this novel major porin, OmpAb, will help in understanding the structure-function relationship.     

The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition

Background

MicroRNAs are modifiers of gene expression, acting to reduce translation through either translational repression or mRNA cleavage. Recently, it has been shown that some microRNAs can act to promote or suppress cell transformation, with miR-17-92 described as the first oncogenic microRNA. The association of miR-17-92 encoded microRNAs with a surprisingly broad range of cancers not only underlines the clinical significance of this locus, but also suggests that miR-17-92 may regulate fundamental biological processes, and for these reasons miR-17-92 has been considered as a therapeutic target.

Results

In this study, we show that miR-17-92 is a cell cycle regulated locus, and ectopic expression of a single microRNA (miR-17-5p) is sufficient to drive a proliferative signal in HEK293T cells. For the first time, we reveal the mechanism behind this response - miR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes involved in the transition between these phases. While both pro- and anti-proliferative genes are targeted by miR-17-5p, pro-proliferative mRNAs are specifically up-regulated by secondary and/or tertiary effects in HEK293T cells.

Conclusion

The miR-17-5p microRNA is able to act as both an oncogene and a tumor suppressor in different cellular contexts; our model of competing positive and negative signals can explain both of these activities. The coordinated suppression of proliferation-inhibitors allows miR-17-5p to efficiently de-couple negative regulators of the MAPK (mitogen activated protein kinase) signaling cascade, promoting growth in HEK293T cells. Additionally, we have demonstrated the utility of a systems biology approach as a unique and rapid approach to uncover microRNA function.     

Preferential binding of quinolones to DNA with alternating G,C / A,T sequences: a spectroscopic study

The binding of quinolones, nalidixic acid (Nal), oxolinic acid (Oxo) with double stranded polynucleotides was undertaken by using UV-melting, UV-Vis absorption, fluorescence and CD spectroscopic techniques. The binding of Nal or Oxo to the polynucleotides under low-salt buffer conditions were determined for poly (dA).(dT), poly [d(A-T)], poly (dG).(dC), poly [d(G-C)] and E. coli DNA. The fluorescence data were analyzed using a previously established two step mechanism with two different DNA-Drug complexes [Rajeswari et al., Biochemistry 26, 6825-31 (1987)]. The first complex [DN](1) with a binding constant K(1), is formed where the interactions are 'nonspecific' and complex [DN](2) with a binding constant K(2), is formed where the interactions are "specific" which involve (additional) hydrophobic type of interactions like 'stacking' of the drug and the overall association constant is represented as K(=K(1)K(2)). The order of binding for Nal and Oxo is: poly [d(G-C)] > poly [d(A- T)] > E. coli > poly (dG).(dC) > poly (dA).(dT). Interaction of quinolones seems to be preferential in the alternating G, C or A, T stretches of DNA than those of non-alternating. Within any alternating or non-alternating in DNA sequences the G, C rich sequences have distinctly greater binding than A, T sequences. The overall association constant data (K) reveal higher binding of Oxo to DNA compared to Nal to any given polynucleotide investigated; which also explains the higher antibacterial potency of Oxo. Changes in the absorption difference spectra and in circular dichroic spectra also manifest these results. As the melting temperatures of the polynucleotides were only marginally raised in presence of the quinolone, we rule out the possibility of 'classical intercalation' of the drug. Amino group of guanine facilitates the binding of quinolones and therefore has the greater binding with the DNA. However, poly (dG).(dC) is known to exist in 'A' conformation which is not adopted by quinolones as in the case of poly (dA).(dT). Present results suggest that Nal or Oxo bind to DNA in a non-classical fashion which is partially stacking in nature.     

Rapid, sequential changes in surface morphology of PC12 pheochromocytoma cells in response to nerve growth factor

The effect of nerve growth factor (NGF), a substance that promotes the differentiation and maintenance of certain neurons, was studied via scanning electron microscopy utilizing the PC12 clonal NGF-responsive pheochromocytoma cell line. After 2-4 d of exposure to NGF, these cells acquire many of the properties of normal sympathic neurons. However, by phase microscopy, no changes are discernible within the first 12-18 h. Since the primary NGF receptor appears to be a membrane receptor, it seemed likely that some of the initial responses to the factor may be surface related. PC12 cells maintained without NGF are round to ovoid and have numerous microvilli and small blebs. After the addition of NGF, there is a rapidly initiated sequential change in the cell surface. Ruffles appear over the dorsal surface of the cells with 1 min, become prominent by 3 min, and almost disappear by 7 min. Microvilli, conversely, disappear as the dorsal ruffles become prominent. Ruffles are seen at the the periphery of cell at 3 min, are prominent on most of the cells by 7 min and are gone by 15 min. The surface remains smooth from 15 min until 45 min when large blebs appear. The large blebs are present on most cells at 2 h and are gone by 4 h. The surface remains relatively smooth until 6-7 h of NGF treatment, when microvilli reappear as small knobs. These microvilli increase in both number and length to cover the cell surface by 10 h. These changes were not observed with other basic proteins, with α-bungarotoxin (which binds specifically to PC12 membranes), and were not affected by an RNA synthesis inhibitor that blocks initiation of neurite outgrowth. Changes in the cell surface architecture appear to be among the earlist NGF responses yet detected and may represent or reflect primary events in the mechanism of the factor’s action.     

A smart bioconjugate of chymotrypsin

alpha-Chymotrypsin was immobilized on Eudragit S-100 via covalent coupling with 93% retention of proteolytic activity. The conjugate behaved as a smart biocatalyst and functioned as a pH-dependent reversibly soluble-insoluble biocatalyst. The pH optimum of chymotrypsin broadened on immobilization, and the immobilized preparation showed better stability at and above pH 6.5 as compared to the free enzyme. The immobilized enzyme showed a slight shift in the temperature optimum and enhanced thermal stability retaining 70% of its original activity after 1 h of exposure to 40 degrees C as compared to the 25% residual activity for the free enzyme under identical conditions. K(m) and V(max) values did not change on immobilization. Also, the immobilized preparation was quite stable to reuse, it retained almost 85% of its original activity even after a fifth precipitation cycle. UV spectroscopy and circular dichroism were used to probe structural changes in the enzyme upon immobilization.     

Comparative proteomics of inner membrane fraction from carbapenem-resistant Acinetobacter baumannii with a reference strain

Acinetobacter baumannii has been identified by the Infectious Diseases Society of America as one of the six pathogens that cause majority of hospital infections. Increased resistance of A.baumannii even to the latest generation of β-lactams like carbapenem is an immediate threat to mankind. As inner-membrane fraction plays a significant role in survival of A.baumannii, we investigated the inner-membrane fraction proteome of carbapenem-resistant strain of A.baumannii using Differential In-Gel Electrophoresis (DIGE) followed by DeCyder, Progenesis and LC-MS/MS analysis. We identified 19 over-expressed and 4 down-regulated proteins (fold change>2, p<0.05) in resistant strain as compared to reference strain. Some of the upregulated proteins in resistant strain and their association with carbapenem resistance in A.baumannii are: i) β-lactamases, AmpC and OXA-51: cleave and inactivate carbapenem ii) metabolic enzymes, ATP synthase, malate dehydrogenase and 2-oxoglutarate dehydrogenase: help in increased energy production for the survival and iii) elongation factor Tu and ribosomal proteins: help in the overall protein production. Further, entry of carbapenem perhaps is limited by controlled production of OmpW and low levels of surface antigen help to evade host defence mechanism in developing resistance in A.baumannii. Present results support a model for the importance of proteins of inner-membrane fraction and their synergistic effect in the mediation of resistance of A.baumannii to carbapenem.